Alpha-Tropomyosin (Alpha TM) is a member of a complex multigene family containing multiple isoforms. The type of TM expressed depends on the developmental stage and physiological state of a particular cell type or tissue, although such information is not published for cardiac muscle during development and hypertrophy. Alpha Alpha-TM is present in adult cardiac and skeletal muscle in small mammals. As a way to understand the significance of the different TM isoforms in normal and pathological states, site-directed mutagenesis of Alpha TM will be carried out to investigate structure-function relationships in this important regulatory protein. A full length cDNA clone of Alpha-TM is under construction. The complete cDNA clone will be expressed in E. coli to produce a non-fusion protein that can be purified for studies in vitro. Site-directed mutagenesis will be used to address specific questions about the function of TM based on the coiled-coil model for its structure: are there discrete actin binding sites? What is the true extent of the troponin binding site? What features of TM structure are important for cooperative interaction with actin and myosin? The conserved and non-conserved regions of known TM sequences will be taken into consideration in selecting sites for mutagenesis, as well as changes in cardiac isoforms in development and hypertrophy, as they become known. Oligonucleotide directed mutagenesis is the method for changing single bases in a known fashion. For local random mutagenesis, sodium bisulfite catalyzed deamination of cytosine will be used in conjunction with oligonucleotides. Assays for mutant proteins will include binding to actin and troponin and assay of cooperative activation of the actomyosin ATPase in the presence and absence of troponin.